Current materials and methods for isolating the species in a given biological sample simply are not sufficient to isolate reliably all of the components of such a mixture. Typically, the dominant molecular species will mask those species present in concentrations less than about one one thousandth of the dominant species. For biological samples, such as blood, two of the most predominant molecular species are albumin and immunoglobulins. Attempts to identify various enzymes, antibodies, proteins, or secondary metabolites that may have relevance as disease markers, or which may be relevant for drug discovery, are complicated by the overall high abundance of albumin and immunoglobulins. As a result, the conventional resolving power, sensitivity, and loading capacity of the two most commonly used analytical techniques (i.e., 2-dimensional electrophoresis (2DE) and mass spectrometry) are limited. For example, the presence of such highly abundant proteins in a sample produces large signals with consequent signal overlap (in 2DE) or signal suppression (in mass spectrometry) of the other species present in the sample, which complicates analysis and undermines any conclusions about the catalog of molecular species present in the sample.
To isolate particular molecules, various separation protocols have been developed. For example, in gel electrophoresis proteins are uniformly coated with a negatively-charged detergent (e.g. SDS) and placed in the middleon one end (origin) of a buffered gel (e.g., polyacrylamide gel) between oppositely charged electrodes. When the electrodes are charged, each of the protein molecules travels toward one of the electrodes, according to their net charge at the pH of the buffered polyacrylamide gel the oppositely charged electrode. The speed, or mobility, at which the protein molecules move through the gel toward the electrodes is largely dependent on the size of the molecule, i.e., smaller molecules move faster through the gel matrix. As a result in the differences in mobility, types of protein molecules can be separated and then isolated based on their size.
A variant to gel electrophoresis is isoelectric focusing, which exploits the fact that the net charge of a protein depends on the environmental pH. Most generally, at acidic pH, proteins are globally positively charged while in alkaline pH they are negatively charged. The pH at which a protein has no net charge is called its isoelectric point (“pI”). Isoelectric focusing is an electrophoresis technique in which proteins move under an electric field through a pH gradient. All proteins migrate towards the cathode or the anode until they encounter a pH identical to their isoelectric point. At this isoelectric point the protein loses its charge and stops moving.
Proteins of different isoelectric points stop at different levels positions and are thus separated for subsequent identification. Accordingly, similarly sized molecules, which may move at similar speeds, can be separated after coming to rest at different pH points, as a result of having different pI values. In addition, there are situations in which migration by the size in a given buffered gel and migration by the isoelectric point are crossed for an enhanced separation of protein species from very complex mixtures; the technique used in this situation is called bidimensional electrophoresis. Unfortunately, migration of proteins within an electrophoresis gel network according to these techniques is a very slow process and is generally unacceptable for preparative purposes.
In response, various additional protocols have been developed which have attempted to increase the rate of separation, while preserving the accuracy by which it is performed. There are many types of devices comprising two or more subcompartments that are separated from each other by septa, e.g., monofilament screens, membranes, gels, filters, fritted discs, and the like (collectively, “membranes”). Generally, these devices are assembled from a plurality of essentially parallel frames or spacers, separated from each other by one or more membranes.
Multi-compartment electrolizerselectrolyzers with isoelectric membranes were introduced for processing large volumes and amounts of proteins to homogeneity. For example, see P. G. Righetti, et al., “Preparative Protein Purification in a Multi-Compartment Electrolyser with Immobiline Membranes,” 475 J. CHROMATOGRAPHY 293-309 (1989); P. G. Righetti, et al., “Preparative Purification of Human Monoclonal Antibody Isoforms in a Multi-Compartment Electrolyser with Immobiline Membranes,” 500 J. CHROMATOGRAPHY 681-696 (1990); P. G. Righetti, et al., “Preparative Electrophoresis with and without Immobilized pH Gradients,” ADVANCES IN ELECTROPHORESIS 159-200 (1992). Based on isoelectric focus, this purification concept progresses under recycling conditions. The protein macro-ions are kept in a reservoir and are continuously passed through an electric field across a multicompartment electrolyzer equipped with zwitterionic membranes.
In this system the protein is always kept in a liquid vein, also called a “channel.” Consequently, the protein is not lost by adsorption onto surfaces, as typically occurs in chromatographic procedures. Rather, the protein is trapped in a chamber that is delimited by two membranes that have pI values encompassing the pI value of the protein to be separated. Thus, by a continuous titration process, all other impurities, either non-isoelectric or having different pI values, are forced to leave the chamber. In the end, the isoelectric/isoionic protein of interest will ultimately be present, as the sole species, in the chamber. It should be recognized, however, that the isoelectric and isoionic points of a protein can differ to some extent in the presence of counterions.
U.S. Pat. No. 4,971,670 describes this process. Isoelectric membranes also are addressed in U.S. Pat. No. 4,243,507. U.S. Pat. No. 5,834,272 describes an immobilization of enzymes that keeps them in solution and, hence, under conditions of homogeneous catalysis. In U.S. Pat. No. 4,362,612, adjoining compartments are functionally designed to adjust to different pH values electrophoretically, thereby separating dissolved proteins according to their isoelectric points. Similar multiple subcompartments devices are described in U.S. Pat. Nos. 4,971,670, 5,173,164, 4,963,236, and 5,087,338. Each of these patents discloses a device, which is comprised of a series of parallel spacers, that are separated from each other by membranes, that provides an essentially parallel array of subcompartments.
Similarly, Amersham Pharmacia markets an IsoPrime filter using a plurality of pI-selective membranes arranged in series. In this device the membranes are arranged in ascending or descending pI-selectivity. As a solution passes through the membranes, molecules having pI values between two consecutive membranes are trapped between the membranes. However, this process takes on the order of hours to complete. Invitrogen, Inc. markets a device, the ZOOM IEF Fractionator, which is substantially similar to the IsoPrime device, but which enables the membranes to be individually replaced. However, like the IsoPrime, the ZOOM IEF Fractionator process takes on the order of hours to complete.
Various other separation protocols include: sub-cellular fractionation (Lopez, M. F., Electrophoresis, 2000, 21:1082-1093; Hochstrasser, D. F., et al., Electrophoresis, 2000, 21:1104-1115; Dreger, M., Mass Spectrometry Reviews, 2003, 22:27-56; Patton, W. F., J. Chromatography B, 1999, 722:203-223; McDonald T. G. et al., Basic Res. Cardiol., 2003, 98:219-227; Patton, W. F., et al., Electrophoresis, 2001, 22:950-959; Gerner C., et al., Mol. & Cellular Proteomics, 2002, 7:528-537), molecular sizing (Issaq, J. H., et al. 2003, Hochstrasser, et al. 2000), ion exchange (Lopez, M. F., 2000, 17), immobilized metal interaction chromatography (“IMAC”) for calcium binding protein (Lopez, M. F., et al., Electrophoresis, 2000, 21:3427-3440) or phospho-proteins (Hunt, D. F., et al., Nat. Biotechnol., 2002, 20:301-305), hydrophobic (Lopez, 2000), heparine (Hochstrasser, et al. 2000) or lectin (Hochstrasser, et al. 2000, Lopez, 2000; Regnier, F., et al., J. Chromatography B, 2001, 752:293-306) affinity chromatography, and liquid chromatography (Issaq, J. H., et al 2002, Hochstrasser, et al. 2000).
Two-dimensional liquid chromatography used for intact protein fractionation, or their trypsic digests, generally uses reverse phase (“RP”) for the second dimension, combined with ion exchange (Yates, J. R., Nature Biotech., 1999, 17:676-682, Unger, K. K., et al., Anal. Chem., 2002, 74:809-820), chromato-focusing (Wall, D., et al., Anal. Chem., 2000, 72:1099-1111), size exclusion (Opiteck, G., Anal. Biochem., 1998, 258:349-361), affinity (Regnier 2001), or another RP (Chicz R., et al., Rapid Commun. in Mass Spectrometry, 2003, 17:909-916) as the first chromatography step.
Unfortunately, multidimensional chromatography in proteomic fractionation generally never exceeds two dimensions due to high number of fractions to manage (pH-adjustment, desalting, re-injection in second dimension) and analyze, especially when a tedious analytical methods method such as 2DE makes the final bottleneck. In addition, a related shortcoming of the prior art is a relative inability to adapt the various devices to a particular separation protocol. For example, if a technician desires to identify 20 different proteins within a sample, a system involving only, e.g., eight separation media may be insufficient. In other words, if a sample contains 20 different proteins that have pI values that incrementally vary by 0.1 pH unit, a device having only eight separation media will fail to separate the proteins sufficiently. As a result, the proteins captured by each of the separation media (e.g., based on pI value) may need to be separated by way of a second separation protocol using the same type of separation (e.g., based on pI value).
What is needed, therefore, is an apparatus and a methodology and an apparatus that address at least one if not more of the deficiencies that afflict conventional practice, as previously described. More particularly, the need exists for an approach for separating molecules, such as proteins, quickly and accurately.